Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

Institute of Biochemistry ETH Zurich Otto Stern Weg 3 8093 Zurich Switzerland

Institute of Molecular Cancer Research University of Zurich Winterthurerstrasse 190 8057 Zurich Switzerland

Institute of Molecular Cancer Research University of Zurich Winterthurerstrasse 190 8057 Zurich Switzerland; Institute of Molecular Genetics of the Czech Academy of Sciences Videnska 1083 142 20 Prague Czech Republic

Institute of Molecular Genetics of the Czech Academy of Sciences Videnska 1083 142 20 Prague Czech Republic

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